1. Field of the Invention
The present invention relates generally to the fields of cell biology and bone metabolism. More particularly, it concerns the use of compositions containing one or more CD40 agonists, such as CD40 ligands and/or agonistic anti-CD40 antibodies, to reduce or prevent cell death or apoptosis, in bone cells, such as osteocytes and osteoblasts. Methods of treating or preventing bone loss, including osteoporosis, and methods of reducing or eliminating the bone loss associated with steroid administration are also included. Further provided are a variety of therapeutic kits and cocktails.
2. Description of Related Art
In the body, bone is remodeled continuously through the process of resorption by osteoclasts, followed by bone formation by osteoblasts. Normally, the activity of these two types of cells is balanced through the action of hormones and other signaling mechanisms. During the resorption phase, sites for remodeling are targeted by osteoclasts that form pits in bone, releasing organic matrix and minerals into the circulation. Resorption at a single site can last as long as about three weeks. As resorption progresses, osteoblasts begin filling in the resorbed region with new mineralized bone.
Peak bone mass is reached at about age 30. After pe tne mass is reached, there is a gradual age-related loss of bone mass in both males and females due to a slight imbalance in resorption and formation. However, as estrogen production declines in women around the time of menopause, bone resorption increases dramatically, which can lead to rapid bone loss. Premenopausal women have been shown to turnover bone at a rate of about one-third to one-half gram of bone per day, while the turnover is double to triple that in early postmenopausal women. Although bone loss can be especially elevated in the five to seven years immediately following menopause, this process continues throughout life. The rate of bone loss can vary dramatically from woman to woman.
Several studies have documented this dramatic increase in bone turnover levels at menopause (Ebeling et al., 1996; Garnero et al., 1996; Prestwood et al., 1994). The results showed that the increase in both the mean and the range of bone resorption values stayed high for more than 20 years beyond menopause, a pattern consistent with bone mass changes over a woman""s lifetime. These studies demonstrated an inverse correlation between bone turnover and bone mass, with high turnover associated with low bone mass.
Postmenopausal bone loss may be due to increased production of cytokines such as TNFxcex1 and interleukin 1 and/or increased osteocyte apoptosis. Estrogen has been shown to be a viability factor for osteocytes in both humans and rats (Tomkinson et al., 1997, 1998). Human bone removed from female patients being treated with gonadotropin releasing hormone showed a higher increase in the proportion of dead osteocytes compared to controls. The same was shown to be true for ovariectamized rates compared to controls (Rickard et al., 1992). Estrogen has been reported to modulate TNFxcex1 production by human osteoblast and peripheral monocytes from women who have undergone ovariectomy and produce increased levels of TNFxcex1 (Pacifici et al., 1991). Delivering both IL-1 receptor antagonist and the soluble TNF receptor completely blocked bone loss due to ovariectomy in mice (Kimble et al., 1995). Therefore, a lack of estrogen can lead to increased production of cytokines responsible for bone loss (Lorenzo, 1996).
In addition to bone loss due to aging and estrogen deficiency, patients of all ages, both sexes, and all races are susceptible to steroid-induced bone loss. Administration of glucocorticoids and steroids is the third most common cause of osteoporosis. Steroid-induced bone loss usually affects the cortical and cancellous bone of the axial skeleton. Between 30% and 50% of individuals taking steroids for more than 6 months will develop osteoporosis. The rate of bone loss is very rapid in the initial year of therapy, with as much as 20% of the bone lost in the first year. Doses exceeding 7.5 mg/day of prednisone can cause significant loss of trabecular bone in most people.
Studies in mice administered glucocorticoids suggests that steroid-induced bone loss is due to decreased bone formation which results from higher numbers of apoptotic/dead osteoclasts and osteoblasts. Lesser numbers of these cells could account for changes seen with glucocorticoid-induced bone disease. A decrease in osteoblast and osteocyte cell number due to death/apoptosis has also been demonstrated in patients who have glucocorticoid-induced osteoporosis (Weinstein et al., 1998).
Despite the current understanding and the considerable amount of research in this area, bone loss and osteoporosis remain significant medical and economic problems. Therefore, methods of reducing or preventing bone loss, for example by reducing or preventing apoptosis of osteocytes and osteoblasts, would represent a significant advance in the art.
The present invention overcomes one or more of these and other shortcomings in the art by providing a range of new treatments by which to reduce bone cell death and bone loss. The invention is broadly based upon the surprising finding that CD40 is expressed on bone cells and that CD40 agonists dramatically reduce bone cell death. The invention thus provides methods, compositions and uses of one or more CD40 agonists, such as CD40 ligands and/or agonistic anti-CD40 antibodies, to reduce or prevent bone cell death or apoptosis, thereby providing new treatments for bone loss associated with a variety of diseases and clinical conditions.
The invention therefore provides methods, compositions and uses in reducing or preventing bone cell death and/or apoptosis, comprising contacting a bone cell, a population of bone cells or a cell population comprising bone cells, with a biologically effective amount of at least a first composition comprising at least a first CD40 agonist, such as a CD40 ligand and/or agonistic anti-CD40 antibody.
The bone cells to be treated by the invention include, but are not limited to, osteoblasts and osteocytes. The CD40 ligands and/or agonists may induce the apoptosis of osteoclasts, inhibit the apoptosis of osteoclasts, or exert no detectable effects on osteoclasts, so long as the overall effect of the CD40 ligand and/or agonist inhibits the apoptosis of osteocytes and/or osteoblasts to a greater extent than osteoclasts, or otherwise produces a net beneficial effect on bone mass, bone density, bone cell number or other parameter indicative of health bone tissue.
All CD40 agonists are suitable for use in the invention, so long as they bind to and activate one or more CD40 receptors on a bone cell. A CD40 agonist that xe2x80x9cbinds to and activatesxe2x80x9d a CD40 receptor a bone cell is a biological or chemical component or agent that stimulates cell signaling via CD40 in such cells. xe2x80x9cCell signalingxe2x80x9d via a CD40 receptor is indicated by the capacity to xe2x80x9ctransducexe2x80x9d a signal, i.e., transmit a biological effect, to the intracellular environment by binding of an agent to an extracellular portion of the receptor. Most preferably, CD40 agonists bind to and activate a CD40 receptor on a bone cell, thereby creating a cell signal that reduces cell death and/or apoptosis in the cell.
Agents that xe2x80x9cstimulatexe2x80x9d cell signaling via CD40 receptors may do so directly or indirectly. Although agents that act directly are generally preferred, agents that indirectly stimulate or activate CD40 receptors may be used, including accessory signaling molecules, co-stimulators and the like, and agents that remove, inactivate or downregulate inhibitors of the CD40 signaling process. Included within this group of CD40 agonists are agents that stimulate or xe2x80x9cupregulatexe2x80x9d the expression of the CD40 receptor on bone cells. Such components will therefore increase the amount of the receptor expressed at the cell surface and available for binding to the natural biological ligand counterpart or exogenously added CD40 ligands.
CD40 agonists that directly stimulate or activate CD40 receptors include the biological ligand counterparts to the receptor. Therefore, in preferred embodiments, the at least a first composition comprises at least a first CD40 ligand. As used herein, the term xe2x80x9cCD40 ligandxe2x80x9d will be understood to include any peptide, polypeptide or protein, or a nucleic acid encoding a peptide, polypeptide or protein, that can bind to and activate one or more CD40 receptors on a bone cell. Thus, CD40 ligands for use in the present invention include, but are not limited to, gp39 peptides, polypeptides, proteins or nucleic acids.
Although human CD40 ligands will be preferred for use in human therapy, CD40 ligands from any species may be used in the invention. For use in other animal species, such as in veterinary embodiments, a species of CD40 ligand matched to the animal being treated will be preferred. Therefore, among the gp39 peptides, polypeptides, proteins or nucleic acids contemplated for use in the present invention are the human, murine, canine, bovine, feline and rat gp39 sequences, as well as other CD40 binding proteins, known in the art and disclosed herein. Exemplary CD40 ligands are those of SEQ ID NO:2, as encoded by SEQ ID NO:1 and the related nucleic acid sequence of SEQ ID NO:5; and those of SEQ ID NO:7, as encoded by SEQ ID NO:6. In preferred aspects of the invention, the at least a first CD40 ligand is at least a first human gp39 peptide, protein or nucleic acid.
A CD40 ligand xe2x80x9cprotein, polypeptide or peptidexe2x80x9d, as used herein, refers to a proteinaceous CD40 ligand component that has sufficient biological activity to be biologically effective. Accordingly, xe2x80x9cCD40 ligandsxe2x80x9d include full-length CD40 ligand proteins and polypeptides and also CD40 ligands that have been subject to non-native processing or biological modification. Such modifications include truncations, extensions, active domains or fragments, fusion proteins, mutants with substantial or sufficient biological activity, peptidomimetics and the like.
Any form of CD40 ligand may be used in the invention, including those isolated and purified from natural sources. CD40 ligands prepared by recombinant expression will often be preferred, i.e., those obtained by expressing a CD40 ligand nucleic acid in a recombinant host cell and collecting the expressed CD40 ligand protein. CD40 ligands prepared by automated peptide synthesis are also included.
In certain embodiments, the CD40 ligand is a soluble gp39 peptide, polypeptide or protein, or at least a first nucleic acid encoding a soluble gp39 peptide, polypeptide or protein.
In preferred embodiments, the at least a first CD40 ligand is at least a first soluble gp39 protein that comprises all, substantially all or most of the extracellular domain. In particular aspects, the extracellular domain of the gp39 protein comprises the human sequence from between about amino acid 47-50 to about amino acid 261 of SEQ ID NO:2 or the corresponding bovine sequence. Sequences of from between about amino acid 47-50 to about amino acid 260 of the corresponding murine, canine, feline or rat sequences may be used, as disclosed in U.S. provisional application Serial No. 60/151,250, filed Aug. 27, 1999, incorporated herein by reference. As used herein, the term xe2x80x9cbetween about amino acid 47-50 to about amino acid 260 or 261xe2x80x9d will be understood to include sequences between amino acid 45, 46, 48, 49, 51 and 52 or so to about amino acid 257, 258, 259, 260 and 261 or so.
xe2x80x9cCD40 ligand nucleic acidsxe2x80x9d are DNA or RNA coding regions that encode, and under conditions appropriate for expression, encode and express any one or more of the biologically active CD40 ligand protein- and polypeptide-based components described above, including full-length proteins and polypeptides, and active variants, fragments and fusions thereof. Recombinant vectors, viral vectors and recombinant viruses are preferred for use in various embodiments, as described in detail herein.
Therefore, in certain aspects of the invention, the at least a first soluble gp39 protein is encoded by the sequence from between about nucleotide 160-169 to about nucleotide 804 of SEQ ID NO:1. Sequences of from between about nucleotide 151-160 to about nucleotide 792 of the corresponding murine sequence; between about nucleotide 153-162 to about nucleotide 797 of the corresponding bovine sequence; between about nucleotide 143-152 to about nucleotide 784 of the corresponding canine sequence; between about nucleotide 143-152 to about nucleotide 784 of the corresponding feline sequence; or between about nucleotide 139-148 to about nucleotide 780 of the corresponding rat sequence; may be used, as disclosed in U.S. provisional application Serial No. 60/151,250, filed Aug. 27, 1999, incorporated herein by reference.
In other aspects of the present invention, the methods, compositions and uses provide the CD40 ligand to the target bone cells via cell-based delivery. As such, the methods, compositions and uses of the invention include recombinant host cells expressing at least a first CD40 ligand or agonist on the surface of the cell.
Particularly preferred embodiments of the invention are those wherein the at least a first CD40 ligand is a gp39 peptide or protein oligomer, including naturally forming gp39 peptide, polypeptide or protein oligomers, as well as gp39 peptides, polypeptides, proteins (and encoding nucleic acids) that comprise an oligomerization sequence. While oligomers such as dimers, trimers and tetramers are preferred in certain aspects of he invention, in other aspects of the invention larger oligomeric structures are contemplated for use, so long as the oligomeric structure retains the ability to bind to and activate one or more CD40 receptor(s) on a bone cell. Exemplary oligomerization sequences include, but are not limited to, leucine zipper or lung surfactant protein D sequences. In preferred aspects of the invention, the oligomerization sequence has the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, with SEQ ID NO:3 being particularly preferred.
In further aspects of the invention, the at least a first CD40 ligand is at least a first gp39 peptide, protein or nucleic acid that comprises a distinct antigenic sequence, exemplified by, but not limited to, antigenic sequences for detection, such as FLAG sequences. Fusion proteins comprising CD40 ligand sequences may also be used.
Other preferred embodiments are those wherein the CD40 agonist is a component other than one based upon the natural, biological ligand. As used herein, the term xe2x80x9cCD40 agonistxe2x80x9d thus includes proteins, polypeptides, peptides, antibodies, small molecules and other agents that bind to and activate a CD40 receptor on a bone cell. Thus, CD40 xe2x80x9cagonistsxe2x80x9d are operationally similar to CD40 xe2x80x9cligandsxe2x80x9d, although the agonists are not limited to derivatives of CD40 ligands, but rather include all operative species irrespective of the underlying molecular structure. Suitable CD40 agonists are known in the art and can now readily be identified in light of the functional assays provided in the present disclosure.
In certain preferred aspects, the xe2x80x9cCD40 agonistxe2x80x9d is at least a first agonistic anti-CD40 antibody, or antigen-binding fragment thereof, including, but not limited to, at least a first scFv, Fv, Fabxe2x80x2, Fab or F(abxe2x80x2)2 antigen-binding region of an anti-CD40 antibody. In certain aspects of the invention, the at least a first CD40 agonist is at least a first human, humanized or part-human chimeric anti-CD40 antibody or antigen-binding fragment thereof. In other aspects, the at least a first CD40 agonist is at least a first anti-CD40 monoclonal antibody, including, but not limited to, the G28-5, mAb89, EA-5 or S2C6 monoclonal antibody, or an antigen-binding fragment thereof.
In light of the present discoveries, one or more CD40 agonists may now be used in all in vitro and in vivo methods of reducing or preventing bone cell death and/or apoptosis. All that is required is to contact a composition comprising bone cells with a biologically effective amount of at least a first composition comprising at least a first CD40 agonist.
Such methods and uses include the addition of the CD40 agonist composition to bone cells in vitro. Accordingly, the invention provides methods and uses in culturing bone cells invitro and in generating artificial bone tissue ex vivo. The methods and uses generally comprise providing a biologically effective amount of at least a first composition comprising at least a first CD40 agonist to an in vitro or ex vivo biological sample that contains a population of bone cells.
Preferred methods, uses and medicaments of the invention are those in which the CD40 agonist compositions are provided to bone cells in vivo, simply by administering the composition to an animal or patient. The invention thus provides methods and uses of reducing or preventing bone cell death and/or apoptosis, reducing or preventing bone loss or mass and, generally, treating animals and patients with various forms of diseases and conditions associated with adverse effects on bone.
These methods and uses of the invention comprise providing to an animal or patient at least a first composition that comprises at least a first CD40 agonist in an amount effective to reduce or prevent bone cell death and/or apoptosis or reduce or prevent bone loss in the animal or patient. This is the meaning of the terms xe2x80x9cbiologically and therapeutically effective amountsxe2x80x9d, as used herein, i.e., amounts effective to reduce or prevent bone cell death and/or apoptosis or to reduce or prevent bone loss or mass when administered to an animal or patient.
The in vivo treatment methods of the invention generally require the administration of pharmaceutically or pharmacologically acceptable formulations of CD40 agonist proteins, nucleic acids, vectors or antibodies. Systemic administration, including intravenous administration, is suitable for use in the invention. More localized delivery to the bone is also contemplated.
Whether CD40 ligands, agonists or antibodies, or combinations thereof, are employed in the methods and uses of the invention, the CD40 agonists may be used alone or in combination. Accordingly, the invention encompasses the use of one, two, three, four, five, six or more CD40 agonists, including a plurality of CD40 agonists. Different CD40 ligands, agonists and/or antibodies may be formulated in combination or separately and used simultaneously or sequentially. One time and repeated uses are contemplated.
The one or more CD40 agonists may also be used in combination with other therapeutic agents. Such combination therapy aspects of the invention generally include the formulation, fabrication and/or use of xe2x80x9cbiologically or therapeutically combined effective amountsxe2x80x9d of CD40 agonists and one or more other therapeutic agents. One, two, three, four, five, six or more other therapeutic agents may be used, including a plurality thereof. Depending on the therapeutic objective, the CD40 agonists and other therapeutic agent(s) may be formulated in combination or separately and administered simultaneously or separately, including in any order of administration.
Exemplary combined therapeutic embodiments include CD40 agonists combined for use with a biologically effective amount of at least a first cytokine, such as IL-4 and/or IL-6. In further embodiments, the CD40 agonists are combined with a biologically effective amount of at least a second, distinct agent used to treat or prevent bone cell death, including second, distinct anti-apoptosis agents. One, two, three, four, five, six or more of such agents may be used, including a plurality thereof.
Second, distinct anti-bone loss agents include anti-apoptosis agents, exemplified by, but not limited to, one or more of transforming growth factor beta (TGF-xcex2), IL-6, estrogen, a bisphosphonate and an agent listed in Table 4. Other second, distinct anti-bone loss agents are anti-osteoporosis agents, exemplified by, but not limited to, selective estrogen receptor modulators, such as tamoxifen or raloxifene, alendronate, calcitonin, calcium, fluoride and/or vitamin D.
A wide range of diseases, disorders and conditions associated with bone loss or damage may be treated by the compositions, kits, formulations, methods, uses and medicaments of the present invention. These include both the treatment and prevention of bone loss in animals and patients, wherein the CD40 agonist compositions administered are given in therapeutically and prophylactically effective amounts, respectively.
In certain aspects of the invention, the bone loss is associated with and/or caused by a disease, including, but not limited to, osteoporosis, osteonecrosis or inflammatory arthritis. In other aspects of the invention, the bone loss is caused by aging, estrogen loss or is associated with surgical intervention. The ability to treat or prevent osteoporosis in animals and patients is a particularly important aspect of the invention, whether using CD40 agonists alone or combination therapy.
As the present invention is generally based upon the surprising finding of CD40 expression on bone cells, the use of CD40 agonists can now be intelligently applied to new group of animals and patients. Accordingly, the invention includes the selection, pre-selection or identification of certain animal or patient groups, such as selecting an animal or patient having or at risk for developing bone cell death or apoptosis, or a disease or condition associated with bone loss. Although confined to only one area of treatment, the identification of an animal or patient not suffering from cancer forms one aspect of the invention. Certain of the selection criteria therefore include the identification of an animal or patient not previously provided with a CD40 agonist for another purpose.
In further aspects of the invention, the bone loss is associated with and/or caused by steroid therapy, i.e., the administration of at least a first steroid to an animal or patient. Animals and patients may again be selected, pre-selected or identified on the basis of having or at being in need of steroid therapy or steroid therapy associated with bone loss. Steroid therapy may be administered to treat a condition or disease in the animal or patient, exemplified by, but not limited to, post-menopausal estrogen loss, estrogen loss due to ovariectomy or total hysterectomy, lupus nephritis, Takayasuds arteritis, Wegeners granulomatosis, anti-glomerular basement membrane nephritis, myositis, scleroderma, idiopathic autoimmune thrombocytopenia, asthma, a chronic obstructive lung disease, nephrotic/nephritic syndrome or even cancer. Steroid therapy may also be used in conjunction with an organ or tissue transplant, such as a bone marrow transplant or a multiple organ transplant. In certain aspects of the invention, the at least a first steroid is administered at a high dose and/or over a long period of time.
The invention thus also provides compositions, kits, formulations, methods, uses and medicaments for application in improved steroid therapy and reduced osteodetrimental steroid therapy. The animal or patient undergoing or in need of steroid therapy is provided with a therapeutically or prophylactically effective amount of at least a first composition comprising at least a first CD40 agonist effective to counteract the side-effects of the steroid, such as reducing the osteodetrimental effects of the steroid. This provides steroid therapy with reduced bone loss, wherein the compositions of the invention are administered in amounts effective to reduce the bone loss associated with the steroid or steroid therapy. The CD40 agonists may be administered before, during or after steroid therapy. When given together with steroids, administration may be sequential or substantially simultaneous.
The present invention is applicable to all animals, particularly humans and valuable or valued animals, such as race horses, pedigree animals, domestic pets, and animals used to produce food for human consumption. In addition to human treatment, exemplary embodiments of the invention therefore include the treatment of horses, dogs, cats and the like; the treatment of cows, pigs, boar, sheep, goat, buffalo, bison, llama, deer, elk, and other large animals, as well as their young, including calves and lambs. The treatment of smaller animals, such as rabbits and hares, is also included. The invention encompasses the treatment of birds, particularly those used to produce food for human consumption, such as chicken, turkey, duck, goose, ostrich, emus, dove, quail, and the like. Bone loss in chickens is a notable economic concern.
Still further embodiments of the invention concern therapeutic kits. The kits comprise, in at least a first suitable container, one or more CD40 agonists in conjunction with written instructions for use in reducing or preventing bone cell death and/or apoptosis or treating bone loss. Pharmaceutical compositions of CD40 agonists are preferred.
Combination therapy kits are also provided, which comprise a combined biologically effective amount of at least a first composition, preferably a pharmaceutically acceptable composition, comprising at least a first CD40 agonist and at least a second, distinct therapeutic agent, such as an anti-bone loss agent or a steroid. In preferred embodiments, the at least a second, distinct anti-bone loss agent is an anti-apoptosis or anti-osteoporosis agent.
In certain aspects, the at least a first CD40 agonist composition and the at least a second, distinct therapeutic agent are comprised within a single composition, preferably a pharmaceutical composition. Exemplary combinations are thus therapeutic cocktails comprising CD40 agonists and/or steroids or distinct anti-bone loss agents. In other embodiments, the CD40 agonist and distinct therapeutic agent compositions are comprised within distinct compositions, preferably pharmaceutical compositions. The kits may also comprise at least a third, fourth, fifth, sixth or a plurality of distinct therapeutic agents, such as steroids or anti-bone loss agents. Combinations of CD40 agonists, distinct anti-apoptosis agents and distinct anti-osteoporosis agents are particularly preferred.
Yet further provided by the present invention is a kit comprising, in at least a first suitable container, a biologically effective amount at least a first steroid and at least a first composition comprising at least a first CD40 agonist. The CD40 agonist is preferably provided in an amount effective to reduce or prevent bone loss associated with administration of the steroid to an animal or patient. Such kits may also comprise at least a second, distinct therapeutic agent, such as another steroid or anti-bone loss agent.
Finally, the invention provides for the use of the CD40 agonist compositions in accordance herewith in the preparation of a variety of medicaments for treating one or more conditions associated with bone loss. Such medicaments include the range of CD40 agonist compositions and combinations described above for the treatment of any such disease, disorder or condition.